Naturally-occurring murine and human killer/cytotoxic (NK) cells and precursor populations have been characterized to study the origin, differentiation, and potential function of these cells. We have compared large granular lymphocytes (LGL) with NK activity isolated from mouse livers with intrathymic (i.t.) precursors (dLyl cells) and bone marrow (BM) which contains precursors for both NK cells and thymocytes. It is evident that dLyl and BM successfully transfer i.t. donor-derived cells but LGL do not. By contrast, LGL or BM injected i.v. can transfer donor-derived LGL, whereas, neither population yields high numbers of donor-derived cells in the liver after i.t. transfer. Culture of normal human bone marrow buffy coat (hBM) cells in the presence of human recombinant interleukin 2 rIL2 demonstrated regeneration of NK activity, LGL and cellular proliferation. These cultured, unselected hBM cells showed surface phenotypes of T cells, NK cells, and myeloid cells. After exhaustive elimination of mature T and NK cells, hBM cultures still generated LGL which formed complexes with K562 targets, cells bearing NK cell surface markers, and cells with NK function; whereas, no OKT3+ cells (i.e. T cells) grew. Thus, differentiation of early NK progenitors is IL2 dependent but independent of T cell differentiation or growth; and, hBM NK progenitors lack all of the following markers: E rosette receptor, T101, Fcg-R, OKM1, OKT10, HLel, and NKH1. The culture-generated NK cells from depleted hBM cells are also distinguishable from precursors for myelocytes and macrophages. A stepwise selection procedure to isolate murine mymphocyte subsets from the spleen has been devised to compare their phenotype and function before and after culture in IL2 (Wiltrout project #Z01 CM 09262-04 LEI). Non-T cells, enriched for asialo GM1-bearing cells, contain the NK activity before culture and are responsible for the lymphokine-activated killer (LAK) activity after IL2 culture. Together, the murine and human data strongly suggest a non-T origin of LAK cells.